Transcriptional regulation of vopT by AphA in Vibrio parahemolyticus

Abstract:

[Objective] To study the transcriptional regulation of vopT by AphA in Vibrio parahemolyticus. [Methods] Total RNAs were extracted from the wide-type (WT) strain and the aphA mutant (ΔaphA). Primer extension assay was employed to detect the transcription start site and the promoter activity of vopT in WT, and that in ΔaphA. Quantitative RT-PCR was also applied to calculate the transcriptional variation of target genes between WT and ΔaphA. The entire promoter region of vopT was cloned into the pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and ΔaphA, respectively, to measure the promoter activity (the β-Galactosidase activity) of vopT in WT and ΔaphA by using the β-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), and the entire promoter region of the target genes was amplified by PCR. Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to target promoter regions in vitro. [Results] Primer extension assay detected only one transcriptional start site located at 86 bp upstream of vopT, whose transcript was negative regulated by AphA in an indirectly manner. Moreover, RT-PCR and EMSA results showed that the transcription of vtrA was also indirectly controlled by AphA. [Conclusion] AphA repressed the transcription of vopT indirectly, and this indirect inhibition was not dependent on VtrA.